Peripheral injection of lipopolysaccharide enhances expression of inflammatory cytokines in murine locus coeruleus: possible role of increased norepinephrine turnover

Cytokines and catecholamines are known to constitute a significant portion of the regulatory neuroimmune networks involved in maintaining homeostasis in the central nervous system (CNS). Although we have already reported an increase in norepinephrine (NE) turnover within the locus coeruleus (LC) at 2 and 4 h after the intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), the implication of this increase remains unclear. In view of evidence that norepinephrine (NE) acts in an anti-inflammatory manner by way of negatively regulating pro-inflammatory cytokine expression, we examined the inflammatory cytokine expression levels in the LC of C3H/HeN mice (male, 8 weeks old) after an i.p. LPS injection. The mRNA expression levels of the genes encoding IL-1beta and TNF-alpha within the LC increased during the first 2 h, and showed two peaks, the first at 4 h and the second lesser one at 15 h after the LPS injection. Microglia, which are one of the major cell types that produce pro-inflammatory cytokines in the CNS, were isolated from mouse neonate brains in order to clarify more precisely the relationship between the changes in NE content and the up-regulation of inflammatory cytokines in the LC. Simultaneous incubation of microglia with LPS and NE enhanced the expression of IL-1beta at both mRNA and protein levels, but reduced the mRNA and protein levels of TNF-alpha. These data support the hypothesis that NE negatively regulates the expression of pro-inflammatory cytokine expression, at least in the case of TNF-alpha, which action could contribute to the observed anti-inflammatory properties of NE. This report, based on the results of both in vivo and in vitro experiments, is the first to suggest a relationship between NE content and cytokine expression levels in the CNS.     

The Persistent Membrane Retention of Desipramine Causes Lasting Inhibition of Norepinephrine Transporter Function

The present study examined the potential membrane retention of desipramine (DMI) following exposures of 293-hNET cells to DMI, and its effect on [3H]NE uptake. Incubation of cells with 500 nM DMI for 1 h or 1 day persistently inhibited the uptake of [3H]NE up to 7 days, despite daily repeated washing of cells with drug-free medium. Uptake inhibition was paralleled by persistent retention of DMI associated with cells, as determined by HPLC and by radiotracer experiments using [3H]DMI. [3H]DMI trapped in membranes was displaceable by the structurally unrelated NET inhibitor, nisoxetine, in a concentration-dependent manner, implying interaction of retained [3H]DMI with the NET. A similar cellular retention was observed following incubation of cells with nisoxetine. The results demonstrate that DMI and nisoxetine are persistently retained in cell membranes, at least partly in association with the NET. The retention and slow diffusion of DMI and nisoxetine from membranes may contribute to their pharmacological and modulatory action on NET.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Down-Regulation of Norepinephrine Transporters on PC12 Cells by Transporter Inhibitors

Abstract: To investigate the regulation of norepinephrine transporters (NETs) in vitro, we measured the binding of the NET-selective ligand [³H]nisoxetine in homogenates of PC12 cells after exposure of intact cells to the NET inhibitor desipramine (DMI). A 3-day exposure of PC12 cells to DMI robustly reduced the B _max, but not the K _D, of [³H]nisoxetine binding to NETs. Reduction of the binding of [³H]nisoxetine was dependent on both the concentration of DMI and the time of exposure to DMI. Reduction of [³H]nisoxetine binding to NETs produced by a 1-day exposure to DMI reverted to preexposure levels 48 h after cessation of DMI exposure. Similar down-regulation of NETs was found when PC12 cells were exposed to another NET-selective drug, nisoxetine, which is structurally unrelated to DMI. In contrast, exposure of cells to the serotonin transporter-selective drug citalopram, or the NET substrate norepinephrine, had no effects on the binding of [³H]nisoxetine to NETs. The down-regulation of NETs was paralleled by a DMI-induced reduction in the uptake of [³H]norepinephrine in intact PC12 cells. It can be inferred from these data that inhibitors of the NET can down-regulate NETs directly, and do so in the absence of changes in the synaptic concentration of norepinephrine.     

Corticosterone administration up‐regulated expression of norepinephrine transporter and dopamine β‐hydroxylase in rat locus coeruleus and its terminal regions

Stress has been reported to activate the locus coeruleus (LC)–noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine β‐hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT‐induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT‐induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T‐maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT‐treated rats. In the open‐field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress‐induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion‐induced depression‐like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.

     

Chronic social defeat up-regulates expression of norepinephrine transporter in rat brains

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. However, the molecular link between chronic stress and noradrenergic neurons remains to be elucidated. In the present study adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD) for 4 weeks. Measurements by in situ hybridization and Western blotting showed that CSD significantly increased mRNA and protein levels of the norepinephrine transporter (NET) in the LC region and NET protein levels in the hippocampus, frontal cortex and amygdala. CSD-induced increases in NET expression were abolished by adrenalectomy or treatment with corticosteroid receptor antagonists, suggesting the involvement of corticosterone and corticosteroid receptors in this upregulation. Furthermore, protein levels of protein kinase A (PKA), protein kinase C (PKC), and phosphorylated cAMP-response element binding (pCREB) protein were significantly reduced in the LC and its terminal regions by the CSD paradigm. Similarly, these reduced protein levels caused by CSD were prevented by adrenalectomy. However, effects of corticosteroid receptor antagonists on CSD-induced down-regulation of PKA, PKC, and pCREB proteins were not consistent. While mifeprestone and spironolactone, either alone or in combination, totally abrogate CSD effects on these protein levels of PKA, PKC and pCREB in the LC and those in the hippocampus, frontal cortex and amygdala, their effects on PKA and PKC in the hippocampus, frontal cortex and amygdala were region-dependent. The present findings indicate a correlation between chronic stress and activation of the noradrenergic system. This correlation and CSD-induced alteration in signal transduction molecules may account for their critical effects on the development of symptoms of major depression.     

Norepinephrine transporter expression in cholinergic sympathetic neurons: differential regulation of membrane and vesicular transporters

Sympathetic neurons that undergo a noradrenergic to cholinergic change in phenotype provide a useful model system to examine the developmental regulation of proteins required to synthesize, store, or remove a particular neurotransmitter. This type of change occurs in the sympathetic sweat gland innervation during development and can be induced in cultured sympathetic neurons by extracts of sweat gland-containing footpads or by leukemia inhibitory factor. Sympathetic neurons initially produce norepinephrine (NE) and contain the vesicular monoamine transporter 2 (VMAT2), which packages NE into vesicles, and the norepinephrine transporter (NET), which removes NE from the synaptic cleft to terminate signaling. We have used a variety of biochemical and molecular techniques to test whether VMAT2 and NET levels decrease in sympathetic neurons which stop producing NE and make acetylcholine. In cultured sympathetic neurons, NET protein and mRNA decreased during the switch to a cholinergic phenotype but VMAT2 mRNA and protein did not decline. NET immunoreactivity disappeared from the developing sweat gland innervation in vivo as it acquired cholinergic properties. Surprisingly, NET simultaneously appeared in sweat gland myoepithelial cells. The presence of NET in myoepithelial cells did not require sympathetic innervation. VMAT2 levels did not decrease as the sweat gland innervation became cholinergic, indicating that NE synthesis and vesicular packaging are not coupled in this system. Thus, production of NE and the transporters required for noradrenergic transmission are not coordinately regulated during cholinergic development.     

Somatodendritic α2-Adrenoceptors in the Locus Coeruleus Are Involved in the In Vivo Modulation of Cortical Noradrenaline Release by the Antidepressant Desipramine

Abstract: The effect of the antidepressant and selective noradrenaline reuptake blocker desipramine (DMI) on noradrenergic transmission was evaluated in vivo by dual-probe microdialysis. DMI (1, 3, and 10 mg/kg, i.p.) dose-dependently increased extracellular levels of noradrenaline (NA) in the locus coeruleus (LC) area. In the cingulate cortex (Cg), DMI (3 and 10 mg/kg, i.p.) also increased NA dialysate, but at the lowest dose (1 mg/kg, i.p.) it decreased NA levels. When the α₂-adrenoceptor antagonist RX821002 (1 µ M ) was perfused in the LC, DMI (1 mg/kg, i.p.) no longer decreased but rather increased NA dialysate in the Cg. In electrophysiological experiments, DMI (1 mg/kg, i.p.) inhibited the firing activity of LC neurons by a mechanism reversed by RX821002. Local DMI (0.01–100 µ M ) into the LC increased concentration-dependently NA levels in the LC and simultaneously decreased NA levels in the Cg. This decrease was abolished by local RX821002 administration into the LC. The results demonstrate in vivo that DMI inhibits NA reuptake at somatodendritic and nerve terminal levels of noradrenergic cells. The increased NA dialysate in the LC inhibits noradrenergic activity, which in part counteracts the effects of DMI on the Cg. The modulation of cortical NA release by activity of DMI at the somatodendritic level is mediated through α₂-adrenoceptors located in the LC.     

Inhibition of Na+,K+-ATPase in Penaeus indicus postlarvae by lead

The plasma membrane/mitochondrial fractions of Penaeus indicus postlarvae contain Mg2+-dependent ATPase, Na+,K+-stimulated ATPase, Na+-stimulated ATPase and K+-stimulated ATPase. The Na+,K+-activated, Mg2+-dependent ATPase was investigated further in relation to different pH and temperature conditions, and at various concentrations of protein, ouabain, ATP and ions in the incubation medium. In vitro and in vivo effects of lead were studied on the enzyme activity. In vitro lead inhibited the enzyme activity in a concentration-dependent manner with an IC50 value of 204.4 microM. In correlation with in vitro studies, in vivo investigations (both concentration and time dependent) of lead also indicated a gradual inhibition in enzyme activity. A maximum decrease of 85.3% was observed at LC50 (7.2 ppm) of lead for concentration-dependent experiments. In time-dependent studies, the decrease was maximal (81.7%) at 30 days of sublethal exposure (1.44 ppm). In addition, the substrate- and ion-dependent kinetics of Na+,K+-ATPase was studied in relation to in vitro exposure of lead; these studies suggest a non-competitive type of inhibition.     

Norepinephrine transporter expression and function in noradrenergic cell differentiations

The classical view of norepinephrine transporter (NET) function is the re-uptake of released norepinephrine (NE) by mature sympathetic neurons and noradrenergic neurons of the locus ceruleus (LC; [1-3]). In this report we review previous data and present new results that show that NET is expressed in the young embryo in a wide range of neuronal and non-neuronal tissues and that NET has additional functions during embryonic development. Sympathetic neurons are derived from neural crest stem cells. Fibroblast growth factor-2 (FGF-2), neurotrophin-3 (NT-3) and transforming growth factor-1 (TGF-1) regulate NET expression in cultured quail neural crest cells by causing an increase in NET mRNA levels. They also promote NET function in both neural crest cells and presumptive noradrenergic cells of the LC. The growth factors are synthesized by the neural crest cells and therefore are likely to have autocrine function. In a subsequent stage of development, NE transport regulates differentiation of noradrenergic neurons in the peripheral nervous system and the LC by promoting expression of tyrosine hydroxylase (TH) and dopamine--hydroxylase (DBH). Conversely, uptake inhibitors, such as the tricyclic antidepressant, desipramine, and the drug of abuse, cocaine, inhibit noradrenergic differentiation in both tissues. Taken together, our data indicate that NET is expressed early in embryonic development, NE transport is involved in regulating expression of the noradrenergic phenotype in the peripheral and central nervous systems, and norepinephrine uptake inhibitors can disturb noradrenergic cell differentiation in the sympathetic ganglion (SG) and LC.     

The effects of thyroxine and methimazole treatment on the synthesis of prostacyclin (PGI2) in the rat

The effects of thyroxine (T4) and methimazole administration on plasma prostacyclin (PGI2) levels in vivo and on PGI2 release by aortic rings incubated in vitro were investigated in rats. Male rats were given single injection of T4 (200 micrograms/100 g body wt) ip every 24 h for either 3, 7 or 14 days for hyperthyroid rats. For hypothyroid rats, a group of rats were given methimazole (0.01 % in drinking water) for 14 days. PGI2 concentrations were determined in plasma and also in the medium in which aortic rings were incubated. PGI2 was measured as 6-keto-PGF1 alpha by RIA. Plasma PGI2 levels in T4-treated groups were found to be significantly higher than those of control animals. Aortic rings obtained from rats given single injection of T4 for 7 and 14 days showed significant increases in release of PGI2 into the incubation medium. In contrast, rats given methimazole for 14 days showed a significant decrease in the production of PGI2 by aortic rings without any significant changes in plasma levels. Direct addition of T4 into the incubation medium did not cause any significant changes in PGI2 release by aortic rings obtained from control rats. These results suggest the regulatory role of thyroid hormone in PGI2 synthesis in vivo.     

Upregulation of myocardial syntaxin1A is associated with an early stage of polymicrobial sepsis

This study was designed to test whether increased sympathetic stimulation during polymicrobial sepsis modulates the profile of the syntaxin1A and norepinephrine transporter (NET) in the heart. Sepsis of mild and severe intensity was induced in male Sprague-Dawley rats (275–350 g) using the cecal inoculum (CI) and cecal ligation and puncture (CLP) methods, respectively. The heart samples were isolated from sham, 1, 3, and 7 day post-sepsis in the CI model and at 2 and 20 h post-sepsis in the CLP model. In the CI model, the plasma concentration of norepinephrine (NE) significantly increased at 1, 3, and 7 days post-CI compared to the sham group. The myocardial syntaxin1A mRNA and protein expression also significantly increased at 1 day post-CI compared to the sham group. However, the sepsis-induced increase in syntaxin1A returned to the baseline values at 3 and 7 days post-CI. The expressions of myocardial NET mRNA and protein were not altered at 1 day post-CI but significantly decreased at 3 days post-CI compared to the sham and 1 day post-CI groups. The immunohistochemical analyses revealed an increased localization of NET and syntaxin1A in the heart tissue sections of the 1 day post-CI group. In the CLP model of severe sepsis, the myocardial syntaxin1A mRNA protein expressions significantly increased at 2 h post-CLP, but remained unchanged at 20 h post-CLP compared to the sham group. In contrast, the myocardial expressions of NET mRNA and protein significantly decreased at both 2 and 20 h post-CLP compared to the sham group. It appears that during severe sepsis (CLP model), both the upregulation of syntaxin1A and the downregulation of NET contribute to increased concentrations of NE during the early and late stages of sepsis.